Project Summary/Abstract Endometriosis affects up to 10% of women of reproductive age often causing painful symptoms and an increased risk of infertility. A major clinical problem is the lack of a noninvasive and early diagnostic procedure. The diagnostic gold standard is laparoscopic surgery and histology of the recovered lesions. The goal of this Phase I project is to develop a noninvasive diagnostic test for endometriosis that focuses on early changes in DNA methylation identified from published biomarker discovery. Assays will be based on the MethylMeter platform. A fragmented DNA sample will be fractionated into a methylated DNA fraction and an unmethylated DNA fraction with the use of magnetic beads bearing a methyl-CpG-binding domain protein. The amount of a targeted biomarker in each fraction will be quantified by Coupled Abscription PCR Signaling (CAPS). CAPS combines target DNA amplification with signal amplification. Amplicons with linked promoters are created during the PCR step. The promoters are then subjected to abortive transcription with an RNA polymerase (Abscriptase) to produce a uniform population of 11 nt long RNAs also known as abscripts. A fluorescent signal is created by each abscript by participating in the opening of a molecular beacon. CAPS is more sensitive than qPCR because each amplicon generates multiple signals as opposed to one. Assays will be developed for methylation detection targeted to specific CpG islands and surrounding sequences. Assay development will involve the identification and validation of PCR primers taking into consideration additional sequence for the linked abortive promoters. PCR conditions will be optimized to eliminate amplification bias against methylated versions of the targets. Conditions for methylated DNA binding will be optimized for a minimum of 5 targets. Phase II will focus on the clinical validation of the individual target assays and the development of a diagnostic signature. Protocols for sample collection and processing also will be developed.